PORPHYRIN AND HEME METABOLISMPorphyrins metal and proteinHemoproteinsHemeHemoglobinIronGlobin chainsProtoporphyrin III (IX)

PORPHYRINSNOMENCLATURETypes of substituentsSymmetry I or IIIOxidation between ringsMethylene -CH2-Methene -CH=

HemeProtoporphyrin III

prefix or suffixring substituentsbetween rings

uro-acetate, propionate--

copro-methyl, propionate--

proto-methyl, propionate, vinyl

-porphyrinogen--methylene

-porphyrin--methene

Reactions forProtoporphyrin IX

Step 1Synthesis of d-amino levulinic acidMitochondrial locationRate limitingPyridoxal phosphate (decarboxylase)Regulation of enzyme levels by iron and protohemin

Step 2Synthesis of porphobilinogenAlso called porphobilinogen synthaseZinc-dependentSite of lead toxicity

Further ReactionsStep 3 Tetrapyrrole formationsynthesis of hydroxymethylbilanesynthesis of uroporphyrinogen IIIStep 4 Conversion to protoporphyrin IIIuro to coprocopro to protoporphyrinogen to porphyrinStep 5 Protoheme synthesisinsertion of ferrous ironsite of lead toxicity

1233445

Heme ProteinsProtoheme (or heme) + globin ~ hemoglobinVariations in hemeFe ligands 4, 5, or 6Ferrous or FerricProtoporphyrin III attachment to protein

Heme bHeme cHeme a

Iron-IRE

PorphyriasTreatmentHematin (hemin hydroxide)

Heme DegradationFig. 44.7Page 839

ReactionsFig. 44.8Page 840

Heme oxygenaseBiliverdin reductaseSerum albuminGSH S-transferaseBilirubin UDP-glucuronyl transferaseSpleen MacrophagesBloodLiver

Heme DegradationFeaturesReactionsJaundicehemolyticobstructiveNeonate kernicterusliver diseaseGilberts diseaseBlood Proteinsserum albuminhaptoglobinhemopexin

Blood So FarPlasmaErythrocyteHemoglobinGlobin chainsProtoporphyrin IIIIron

Iron Balance

IRON METABOLISMFig. 44.6Page 838

Iron AbsorptionLow but regulatedFerrous iron conversion needed Heme iron by separate pathwayReducing agents aid uptake-vitamin CFactors in breast milk facilitate uptake (lactoferrin)

Promoters and inhibitors of non-heme iron absorptionPromoters:Ascorbic acidMeatCitric AcidSome spices-caroteneAlcoholInhibitors:Phytic acidPolyphenolsTanninsCalcium

Adapated from Paul Sharp Kings College UK

*Heme synthesis issues: porphyriasHeme breakdown issues: jaundice

Iron in the hemoglobin structure can be reused. The heme group is removed after use. This is referred to as protoporphyrin III (IX). *Synthesis starts with the formation of a pyrrole ring. Two double bonds and a nitrogen. These are derived from two common precursors: succinate (part of Krebs cycle) and glycine.

When we assemble this, we take four of these rings and make a large organic structure with iron sitting in the center. 8 different substituent possibilities. But only three actually attach to the Cs opposite the N. If the groups attached to the outside of the ring are symmetrical, this is Type I symmetry, Type III is asymmetrical. The four pyrrole rings are linked by a carbon link. Different names for a methylene or methene connection. *In this case, there are three different side groups.

Type I and Type III difference in this case there arent evenly arranged CH3. Always single or double ONLY connections. *Succinate (activated as a CoA ester) and glycine starts things off. The final product, heme, has a predecessor called protoporphyrin IX. Why isnt this III? When this work was first begun and they realized that the heme group had three substituents, they didnt know how they were arranged. A chemist drew out all possibilities and the ninth one he drew was the right one. Thus the IX. At right are the rare, inborn errors of metabolism that can happen at each of these steps.

Here: the final product is a feedback inhibitor for the first step in the process. If final product is not produced, the process keeps pumping along. But in these porphyrias, the wrong stuff is produced and builds up. Nastiness follow. Could be behavioural, too. Color urine, skin lesions.

Most common: porphyria cutanea tarda. They get blisters, may be sensitive to UV light, have prolific hair growth. Werewolf legend: may be a person who had porphyria.

*Heme synthesis occurs in bone marrow. Thats where reticulocytes arise so this is logical. Cells need good, healthy mitochondria for this process to begin. Glycine comes in through a transporter or can be derived from serine.

Need PLP as a cofactor. Rate limiting based on whether or not the protein is there. There is a fast turnover (minutes) the body is always making ALA when we need it and not making it when we do not.

PLP- metabolism facilitates decarboxylation in formation of deltaaminolevulinic acid.

Regulated by how much iron is in the cells as well as by how much protohemin end product is about. Fe stimulates synthesis of this. Protohemin: slows/shuts down this cellular machinery. *Next step: take two delta-ALA and condense them. Called delta-ALA dehydratase (removes two waters). It relies on Zn. This forms the actual pyrrole ring. Two things stick out acetate and proprionate. Also, there is a methylene carbon and a nitro group. Porphobilinogen.

Pb toxicity kills this step.

Next: four porphobilinogens are connected. *Uro = only acetate and propionate side groups. Done in cytoplasm.Uro-copro - ?Copro-proto: get vinyl groups. After the vinyl groups added, back to mitos. Then the four carbons that hold stuff together are oxidized and the iron is added to make heme from protoheme. Second lead-tox site is where an enzyme adds the iron. *Lead poisoning: get delta-aminolevulinic acid buildup. If you see this, there is possibly lead poisoning. ALA cannot condense and spills out into the urine.

If all works well, though, we get porphobilinogen. Uroporphyrinogen synthase: allows for closure of the ring. Symmetry switch at this point.

Two-groups = copro. Then to mitos.

If more protoheme is needed than is needed for heme synthesis, it accumulates. And then some bad stuff happens.

Ferrochelatase adds the iron, forming protoheme. Protoheme from heme is that protoheme is not yet associated with any kind of a protein (myoglobin, cytochrome, hemoglobin). *If too much protoheme is there, sometimes an electron will go from iron to iron III, forming superoxide. Product of this is protohemin. Hemin contains Fe+3. Theres an excess of heme biosynthesis going. This (hemin or protohemin) is our feedback inhibitor

Heme is used in lots of places and in many variations. In protoheme, theres nothing other than four pyrrole rings around the iron. But other things have different attachments. Cyt C = all six positions are always fixed. Some cases have a +2 (ferrous) or +3 (ferric) iron. Some cases the side chains have covalently attached side chains, sometimes they just are hydrophobically attached to a pocket. *Cyt. C = methionine reside attaches the heme to a protein.Cyt. Oxidase; heme A has a different connection. *Iron-IRE (iron regulatory element). In 5 untranslated region of the gene, iron binding protein binding to this determines whether or not this is translated. Negatively regulated by protohemin accumulation. It represses transcription of the delta-ALA synthase gene. But Iron-IRE stimulates its formation.

Protohemin disinhibits formation of globin from amino acids.

Hydroxymethylbilane synthase (takes single pyrrole to tetrapyrrole) is regulated by production of erythropoietin. This comes from the kidney. People with renal dysfunction have severe anemia due to loss of erythropoietin. So it is give as a supplement at times. *How would you treat somebody with this issue? Maybe treat with the feedback product. Lesch-Nyhan: failure to use purines properly. To treat, give something that stops biosynthesis of purines. In this case, give hematin (hemin hydroxide) to slow ALA synthase and the entire pathway.

Colored boxes: one step removed from one another yet the phenotypes are crazily different. Red: irritated skin. Green: from CNS stuff skipped for now. *After 120 days, the RBC bites the dust. Taken out of circulation at the spleen. This is the way we want to get rid of RBCs if we dont want craziness to follow. Otherwise Fenton chemistry could occur.

Bilirubin complexes with albumin, which is further metabolized in the LIVER. Liver disease there are issues metabolizing heme. Next step: bilirubin diglucuronide: solubilized. This chemical is stored in the gall bladder and excreted into the gut. Thus goeth the heme. Microorganisms in the intestines take this product, fiddle with it andSome pigments are reabsorbed back into the blood supply and lost through the urine. This gives our urine color. Dark brown of feces? Derived from heme breakdown. Jaundice: yellow from buildup of heme. *ONLY PLACE WE ACTUALLY MAKE CO IN METABOLISM!!!!!!

Next: linear tetrapyrrole. Very highly pigmented (dark green). Biliverdin reductase uses NADPH to produce bilirubin. Bilirubin: not as conjugated, more orangey-brown. *Albumin/bilirubin complex percolates along, reaches the liver, and a series of abundant glutathione-S-transferases act upon the complex. 4-5% of total protein in a liver. VERY abundant. There are ~7 different genes that code for these proteins. Around as homodimers. Two binding sites: hydrophobic things and heme-related stuff. If the bound thing can bind the glutathione, they bond and form an adduct. This is akin to leukotriene biosynthesis. This is another pathway to get rid of drugs. Bromobenzene would form an adduct, the bromine would be displaced, and detox would follow. Their endogenous nature would be, maybe, healthy liver removing hydrophobic compounds from the blood. Years back, to check liver function something was added to the blood and it was seen how fast the liver cleared it via this pathway. But the MAIN goal of this is to remove bilirubin from albumin and bring it into the hepatocyte.

Spleen macrophages produce biliverdin, then bilirubin.Blood- transport w/ albumin to liver.Liver: GSH-transferase sequesters it from blood, and other enzyme conjugates to diglucuronide for removal. Liver not working? Theres an issue with bilirubin metabolism. Now we need to make it more water soluble. We take hydrophobic bilirubin, and add polar groups to it with B-UDP-G-T. Then it is shipped to the gall bladder and released from there. Tis a diglucuronide. Not ALL of it goes to the gall bladder. Indirect reacting fraction: ?????????????????*Hemolytic: RBCs bust prematurely. Could be Vit. E deficiency. Obstructive: spleen and liver work, but gall bladder cannot empty. Cholestasis. Diglucuronide = due to block, not from failure of excretion. One of few cases where there would be high diglucronide. Indirect reacting: albumin-associatedDirect reacting: free? Some believe that bilirubin is a potent antioxidant. But in infants, it screws with CNS function. Neonatal jaundice: caused by one of two things lack of UDP glucuronyl transferase or, more importantly, the fact that neonates have a very high cells (polycythemia) and have PILES of RBCs switching from fetal to adult hemoglobin. Normally treated by UV light, which breaks up some of the bilirubin, making it more soluble. Gilberts disease: genetic, high fasting bilirubin. Due to a limitation in UDP-glucuronyl transferase. Will show up as a possible liver problem but dont actually have one. Crigler-Nager: more sever forms of this Gilberts disease. In one case, UDP-glucuronyl transferase is absent. Other form is partial loss of this. Blood proteins: serum albumin. Critical role in delivering bilirubin to the liver for metabolism. Indirect fraction of bilirubin because you have to extract it inorder to get it it. But the glucuronide reacts directly. Albumin can also bind the heme group if it were to escape, somehow. The Fe would spontaneously oxidize to +3. Must be inisde of cell in a high reducing environment to remain as +2. Binds both bilirubin and hemin.Haptoglobin: alpha 2 globulin, an acute phase reactant, Lots of IL-6 raise this crazily. Important protein for protecting in event that an RBC breaks open. As soon as hemoglobin present in a RBC is exposed to environment, it forms methemoglobin. Haptoglobin binds methemoglobin readily. And macrophages of liver degrade this stuff. Hemopexin: like albumin, is able to bind the hemin group. Only hemopexin and hemin bind even tighter. If heme group gets away from protein, this stuff is able to grab right onto that hemin where there is a hemopexin receptor which clears the hemin from circulation. Protects against Fenton issue. **We have ~4 gm Fe as males, 2.5 grams in females. There are two iron pools. Non-heme (all but heme) and the heme pool (MOSTLY associated with hemoglobin). Stored in hepatocytes and Kupffer cells. Males typically have 1 g. of Fe in the liver, but females have ~400 mg. There is some in muscle in the cytochromes and myoglobin (but is not mobile). *From textbook. To be absorbed, must be absorbed through mucosa of the gut this is highly regulated. It is bound to transferrin in the blood and is the major plasma iron protein. It delivers Fe to cells that need iron. Transferrin receptor is upregulated when there is an immune response. Transferring also delivers iron to the marrow for hematopoiesis.

If 4 g of Fe in an adult male, there is very, very little in the plasma at any given time. *We regulate iron level mainly at the level of iron absorption. Other than phlebotomy, we cannot really get rid of it. Other metals (Cu, Zn) have a bile cycle that keeps stuff around. This isnt true for Fe, and thus we only want to absorb it when we need it. An intricate mechanism controls this.

Daily Fe absorption: 10% of the iron consumed. Chromium we only absorb 1% of what we take in; this is not because of regulation, however thats inherent. But iron taken in is very carefully regulated.

Things that favor formation of Iron II facilitate uptake. We also take up heme iron in a separate, unidentified pathway. Heme iron is more bioavailable than free iron.

Vitamin C (reducing agent) favors ferrous iron and more uptake. During the life cycle, there are important exceptions to low uptake of iron. Babies can absorb lactoferrin readily. Greater bioavailability. These lactoferrin receptors disappear. Last two trimesters of pregnancy the mother ups her iron uptake greatly so that the baby can form.

*Phytic acid is also a zinc antagonist. In some cultures with a bunch of phytate in the dies, they are anemic. Tannins in tea.